Caltractin, a calcium-binding protein (CaBP) related to calmodulin, has been localized to the microtubule-organizing centers (MTOCs) in several eukaryotic systems including human cells. Caltractin is required for normal MTOC duplication and separation, and is of considerable interest due to its implicated role in the cell cycle. Caltractin belongs to the EF-hand superfamily of CaBPs, which maintain high sequence similarity while exhibiting a wide range of Ca2+-binding affinities, presumably leading to their functional diversity. Caltractin displays distinct functional, physical and spectroscopic properties not shared by other members of the EF-hand superfamily. The distinct spectral properties and unusual stability in the apo state make caltractin an excellent candidate for high resolution structure determination by NMR. Moreover, caltractin is phosphorylated in vivo, further distinguishing it among related CaBPs. In the proposed project, 15/N- and 13/C-labeled caltractin samples will be overexpressed and purified. Initial NMR studies will focus on apo caltractin and calcium-loaded caltractin, both in the unphosphorylated and phosphorylated states. Comparative analysis will reveal which states to proceed with full structure determinations. Comparative analysis of the high resolution structures obtained will be performed with available structures of related CaBPs. This analysis will provide valuable new insight into the molecular basis for calcium binding affinity and specificity, and will potentially fill a critical gap in our understanding of the effects of phosphorylation on the structure of a protein, and the role of phosphorylation in the cell cycle.